1. Field of the Invention
This invention relates to a bacterial culture having the ability to metabolize the nitrogen-containing heterocycle carbazole and a method for removing nitrogen from nitrogen-containing solid and liquid hydrocarbon and carbonaceous materials. More particularly, this invention relates to the use of a culture of Sphigomonas sp. having the capability of expressing a carbazole degradation trait constitutively for the removal of carbazole from solid and liquid hydrocarbon and carbonaceous materials including, but not limited to, shale oil, crude oil, petroleum products, coal tar and mixtures thereof and for the selective cleavage of C—N bonds in such solid and liquid hydrocarbon and carbonaceous materials.
2. Description of Related Art
Heavy crude oils and residuum constitute a significant and ever-increasing portion of the world petroleum reserves. These heavy oils possess high calorific content yet have comparatively low market values chiefly because of high sulfur, nitrogen and metals content, high viscosity and molecular weight. The presence of nitrogen-containing compounds and associated metals in petroleum can contribute to inactivation of catalysts in hydrotreating and catalytic cracking processes, thereby causing a decreased efficiency of these refinery operations. The problems associated with heavy oils have prompted the preferential utilization of light crude oils. As light crude oils are consumed at a disproporitonal rate, the amount of heavy oil as a percentage of remaining or world petroleum reserves continues to escalate.
New technologies capable of dealing with heavy oils to mitigate environmental concerns and to allow processing in conventional refineries in a cost-effective manner are needed. Biorefining is one such technique.
Carbazole is a nitrogen-containing heterocycle that is one of the main components of shale oil, crude oil, petroleum products and coal tar. In addition, carbazole is frequently used in the production of dyes, medicines and plastics. The combustion of crude oil transforms nitrogen therein into nitrogen oxides which, when released into the atmosphere, contribute to the formation of acid rain. Nitrogen compounds and associated metals in petroleum can also deactivate catalysts used in oil refineries and can contribute to the chemical instability of refined petroleum products. Accordingly, there is a need for methods for removing nitrogen from petroleum in order to protect the environment and to allow refineries to maximize efficiency.
A variety of bacterial cultures that possess the ability to selectively remove sulfur from organosulfur compounds such as dibenzothiophene have been described and a biochemical pathway for the selective cleavage of carbon-sulfur bonds is known. The use of biodesulfurization to selectively remove sulfur from petroleum and coal while retaining the fuel value has been demonstrated. No such similar pathway is known for the selective cleavage of carbon-nitrogen bonds.
A variety of carbazole-degrading microorganisms are known to exist including Sphigomonas CDH-7, Sphingomonas sp. CB3, Pseudomonas resinovorans CA10, Pseudomonas CA06, Pseudomonas OM1, Pseudomonas LD2, Mycobacterium, Ralstonia, Xanthamonas and Sphigomonas KA1. To the extent that these differing species of carbazole degraders have been studied, it appears as if they all follow a similar carbazole degradation pathway that begins with the oxidative cleavage of the hetercyclic nitrogen ring of carbazole to form 2-aminobiphenyl-2,3-diol. Thus, the initial step in the metabolism of carbazole, catalyzed by carbazole 1, 9a-dioxygenase (CARDO), results in the cleavage of one of the two carbon-nitrogen bonds. However, subsequent biodegradation of carbazole by all cultures characterized heretofore involves degradation of one of the aromatic rings. Most carbazole degrading cultures produce both 2-hydroxypenta-2,4-dienoate and 2-aminobenzoate (anthranilic acid). 2-aminobenzoate undergoes cleavage of the amino substituent to yield catechol that subsequently enters the tricarboxylic acid cycle.
Some carbazole-degrading cultures, like Sphigomonas sp. CB3, have been found to contain carbazole dioxygenases that are related to biphenyl oxidases while other cultures, such as P. resinovorans CA10 (GenBank Accession No. D89064), contain carbazole dioxygenases that show no close relationship to other characterized oxidases (Shepherd, J. M. et al., “Novel Carbazole Degradation Genes in Sphigomonas CB3: Sequence Analysis, Transcription, and Molecular Ecology”, Biochemical and Biophysical Research Communications, 247, 129-135 (1998)). CARDO consists of three components: a dioxygenase, ferredoxin, and ferredoxin reductase. The genes encoding CARDO in Sphigomonas sp. CB3 are contiguous, while in P. resinovorans CA10 they are not. The dioxygenase from Sphingomonas sp. CB3 consists of two subunits, as contrasted with the single subunit present in P. resinovorans CA10. Additionally, the carbazole dioxygenase enzyme of Sphigomonas sp. CB3 has a rather narrow substrate range and does not metabolize naphthalene, dibenzothiophene, phenanthrene or fluorene, unlike P. resinovorans CA10 (Nojiri, H. et al., Diverse Oxygenations Catalyzed by Carbazole 1,9a-Dioxygenase from Pseudomonas sp. Strain CA 10”, Journal of Bacteriology, Vol. 181, No. 10, pp. 3105-3113 (May 1999)). The carbazole-degradation phenotype of previously characterized cultures is induced when carbazole is added to the growth medium and genes responsible for anthranilate conversion to catechol are suggested to be induced by carbazole as well (Shepherd, J. M. et al., supra; Nojiri, H. et al., “Organization and Transcriptional Characterization of Catechol Degradation Genes Involved in Carbazole Degradation by Pseudomonas resinovorans Strain CA10”, Biosci. Biotechnol. Biochem., 66 (4), 897-901, (2002); Kirimura, K. et al., “Selective and Continuous Degradation of Carbazole Contained in Petroleum Oil by Resting Cells of Sphigomonas sp. CDH-7”, Biosci. Biotechnol. Biochem., 63 (9), 1563-1568 (1999); Habe, H. et al., “Sphigomonas sp. Strain KA1, Carrying a Carbazole Dioxygenase Gene Homologue, Degrades Chlorinated Dibenzo-p-dioxins in Soil”, FEMS Microbiology Letters, 211, 43-49 (2002)).